Christiane Delage

Antioxidant, anti-inflammatory and antiproliferative properties of sixteen water plant extracts used in the Limousin countryside as herbal teas

The present paper presents the antioxidant, anti-inflammatory and antiproliferative capabilities of 16 plants. These plants can be found in the Limousin countryside and most of them are used in popular medicine as herbal tea. The biological properties of the water-soluble fractions were measured. Antioxidant properties were evaluated by the ESR method in order to visualize the inhibition of the DPPH, superoxide and hydroxyl radicals. Some extracts were good antioxidants by comparison with reference molecules, e.g. vitamin E and quercetin. Antioxidant effects were correlated with the total amount of phenolic compounds contained in the extracts. Also measured were the anti-inflammatory activities of the 16 water-soluble fractions, by evaluating inhibition of lipoxygenase activity. Finally the effects of these plants on the proliferation of melanoma B16 cells were studied.

Ursolic acid induces apoptosis through mitochondrial intrinsic pathway and caspase-3 activation in M4Beu melanoma cells

Over the coming years, skin cancer could become a significant public health problem. Previous results indicate that ursolic acid (UA), a pentacyclic triterpene acid, has pleiotropic biologic activities such as antiinflammatory and antiproliferative activities on cancer cells. As UA represents a promising chemical entity for the protection of human skin, in agreement with tests done by the cosmetic industry, we investigated its effects on the M4Beu human melanoma cell line. In this report, we demonstrated for the first time that UA had a significant antiproliferative effect on M4Beu, associated with the induction of an apoptotic process, characterized by caspase‐3 activation, the downstream central effector of apoptosis. We demonstrated that UA‐induced apoptosis was dependent on the mitochondrial intrinsic pathway, as shown by transmembrane potential collapse (ΔΨm) and by alteration of the Bax‐Bcl‐2 balance, with a concomitant increase in Bax expression and decrease in Bcl‐2 expression. We also showed that UA‐induced ΔΨm was associated with apoptosis‐inducing factor leakage from mitochondria. Taken together, our results suggest that UA‐induced apoptosis on M4Beu cells is accomplished via triggering of mitochondrial pathway. In conclusion, UA could be an encouraging compound in the treatment or prevention of skin cancer and may represent a new promising anticancer agent in the treatment of melanoma.

Effects of extracellular calcium on the growth-differentiation switch in immortalized keratinocyte HaCaT cells compared with normal human keratinocytes.

Abstract:  The keratinocyte growth and differentiation switch, tightly regulated by several mechanisms, is generally associated with decreased proliferation, cell cycle arrest in G0/G1 phase and expression of epidermal differentiation markers, such as keratin 1 (K1), keratin 10 (K10) and involucrin. In vitro, the spontaneously immortalized human keratinocyte cell line HaCaT is often used as a model to study keratinocyte functions. Comparative differentiation studies between HaCaT cells and normal human keratinocytes (NHK) over an extended time‐period have rarely been reported. Therefore, we studied their switch from a proliferating to a differentiated state over 13 days. As culture conditions involved changes in cellular responses, cells were cultured in a specific medium for keratinocyte growth and differentiation was induced by increasing extracellular calcium concentration from 0.09 to 1.2 mm. In NHK, addition of calcium‐induced morphological changes and concomitant decreased proliferation. For HaCaT cells, calcium addition resulted in morphological changes, but in an unexpected manner, cells were more proliferative than when cultured at low calcium levels. HaCaT cell hyperproliferation correlated with cell cycle analysis, showing an accumulation in S/G2‐M phases. Furthermore, RT‐PCR and western blot analysis revealed a delay in the expression of the differentiation markers K1, K10 and involucrin in HaCaT cells compared with NHK. In conclusion, even though calcium‐induced differentiation was not associated with a decreased cell proliferation, HaCaT cells conserved properties characteristic of differentiation.

Characterization of ursolic acid as a lipoxygenase and cyclooxygenase inhibitor using macrophages, platelets and differentiated HL60 leukemic cells.

A new property of ursolic acid, lipoxygenase and cyclooxygenase inhibition, has been described in an acetone‐extract of heather flowers (Calluna vulgaris) wllich could help explain the anti‐inflammatory characteristics of this plant. In mouse peritoneal macrophages, human platelets and differentiated HL60 leukemic cells, ursolic acid, at 1 μM, blocks arachidonate metabolism.

Inhibition of lipoxygenase activity and HL60 leukemic cell proliferation by ursolic acid isolated from heather flowers (Calluna vulgaris)

A compound was isolated and purified from heather flowers (Calluna vulgaris) based on its ability to inhibit lipoxygenase activity. This molecule was characterized as ursolic acid by GC-MS. Ursolic acid was found to be an inhibitor of both potato tuber 5-lipoxygenase and soybean 15-lipoxygenase with IC50 values of 0.3 mM. Ursolic acid also inhibits lipoxygenase activity in mouse peritoneal macrophages at 1 microM and HL60 leukemic cells growth (IC50 = 0.85 microM) as well as their DNA synthesis (IC50 = 1 microM). The possible role of lipoxygenase inhibition in the proliferation of leukemic cells is discussed.

The P2Y2/Src/p38/COX-2 pathway is involved in the resistance to ursolic acid-induced apoptosis in colorectal and prostate cancer cells

One of the hallmarks of cancer is resistance to apoptosis. Elucidating the mechanisms of how cancer cells evade or delay apoptosis should lead to novel therapeutic strategies. Previously, we showed that HT-29 colorectal cancer cells undergoing apoptosis overexpressed cyclooxygenase-2 (COX-2), in a p38 dependent pathway, to delay ursolic acid-induced apoptosis. Here, we focused on elucidating the upstream signaling pathways regulating this resistance mechanism. The role of ATP as an extracellular signaling molecule took a long time to be accepted. In recent years, ATP and its analogs, via the activation of specific purinergic receptors, have been implicated in many biological processes including cell proliferation, differentiation and apoptosis. In the present report, we have demonstrated a novel role involving purinergic receptors and particularly the P2Y(2) receptor in resistance to ursolic acid-induced apoptosis in both colorectal HT-29 and prostate DU145 cancer cells. We found that ursolic acid induced an increase in intracellular ATP and P2Y(2) transcript levels. Upon activation, P2Y(2) activated Src which in turn phosphorylated p38 leading to COX-2 overexpression which induced resistance to apoptosis in both HT-29 and DU145 cells. Furthermore, Ca(2+)-independent PLA(2) (iPLA(2)) and Ca(2+)-dependent secretory PLA(2) (sPLA(2)) were responsible for arachidonic acid release, the substrate of COX-2. Our findings document that apoptosis triggering was dependent on protein kinase C (PKC) activation in both cell lines after ursolic acid treatment.

HT-29 colorectal cancer cells undergoing apoptosis overexpress COX-2 to delay ursolic acid-induced cell death

Colorectal cancer is one of the most common cancer types and the third leading cause of cancer-related death in the western world. Generally, colorectal cancers are resistant to anticancer drugs. Several lines of evidence support a critical role for cyclooxygenase-2 (COX-2) during colorectal tumorigenesis and its role in chemoresistance. In this study, we focused our interest on the role played by COX-2 in apoptosis induced in HT-29 human colorectal cancer cells by ursolic acid (UA), a triterpenoid found in a large variety of plants. We showed that UA-induced apoptosis and that COX-2 was overexpressed only in apoptotic cells. We demonstrated that this overexpression was mediated by the p38 MAP kinase pathway as inhibiting its activation using a p38-specific inhibitor, SB 203580, abrogated COX-2 expression. Inhibiting COX-2 expression either by using a p38-specific inhibitor or COX-2-specific siRNA increased apoptosis. These results demonstrated that COX-2 was involved in a resistance mechanism to UA-induced apoptosis in HT-29 cells. Cells undergoing apoptosis were able to trigger a resistance mechanism by overexpressing a protein such as COX-2 to delay their death. Furthermore, we demonstrated that this resistance mechanism was independent of PGE(2) production as the addition of the specific COX-2 activity inhibitor, NS-398, did not affect apoptosis in UA-treated cells.

Quercetin 3-[triacetylarabinosyl(1→6)galactoside] and chromones from Calluna vulgaris☆

The new quercetin 3-[2,3,4-triacetyl-alpha-L-arabinopyranosyl (1-->6)-beta-D-galactoside] has been isolated along with 5,7-dihydroxychromone and 5,7-dihydroxychromone 7-beta-D-glucoside from fresh flowers of Calluna vulgaris. Structural elucidation of these natural products was achieved mainly by 1H and 13C NMR.

Phenylpropanoids from leaves of Juniperus phoenicea

Abstract Two new compounds, junipetriolosides A (3-methoxy-4-hydroxy-phenylpropane-7.8-(2′,1′- O -β- d -glucopyranosyl)-7.8,9-triol) and B (3-methoxy-4- O -β- d -glucopyranosyl-phenylpropane-7,8,9-triol) have been isolated from a methanolic extract of the aerial parts of Juniperus phoenicea , along with the rare compound, guaiacylglycerol (3-methoxy-4-hydroxy-phenylpropane-7,8,9,-triol). Structural elucidation of these natural products was achieved mainly by spectroscopic methods.

A novel form of melanoma apoptosis resistance: Melanogenesis up-regulation in apoptotic B16-F0 cells delays ursolic acid-triggered cell death

Melanoma is one of the most aggressive forms of cancer with a continuously growing incidence worldwide and is usually resistant to chemotherapy agents, which is due in part to a strong resistance to apoptosis. The resistance mechanisms are complex and melanoma cells may have diverse possibilities for regulating apoptosis to generate apoptotic deficiencies. In this study, we investigated the relationship between melanogenesis and resistance to apoptosis induced by ursolic acid, a natural chemopreventive agent, in B16-F0 melanoma cells. We demonstrated that cells undergoing apoptosis are able to delay their own death. It appeared that tyrosinase and TRP-1 up-regulation in apoptotic cells and the subsequent production of melanin were clearly implicated in an apoptosis resistance mechanism; while TRP-2, a well known mediator of melanoma resistance to cell death, was repressed. Our results confirm the difficulty of treating melanomas, since, even undergoing apoptosis, cells are nevertheless able to trigger a resistance mechanism to delay death.